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Title:
HYBRID CAR AND HYBRID TCR CELLS TO TREAT INFECTIOUS DISEASES
Document Type and Number:
WIPO Patent Application WO/2021/180832
Kind Code:
A1
Abstract:
The present invention relates to the field of controlling infections where pathogens can be targeted at the host cell-associated and cell-free stages of their life cycles with edited T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR) cells which further secrete pathogen- specific antibodies. An example of such pathogen is human immunodeficiency virus (HIV). Indeed, the present invention discloses that CAR cells producing broadly neutralizing anti-HIV antibodies are capable to both destroy HIV-infected cells and non-cell associated HIV particles that become released from HIV-infected cells in the productive stage of infection. CAR- dependent killing function combined with simultaneous secretion of soluble antibody mediating neutralization of pathogen, antibody-FC fragment mediated cellular cytotoxicity and endocytosis by antigen presenting cells following opsonization present a multiplexed approach for treatment of different infections.

Inventors:
WITKOWSKI WOJCIECH (BE)
VANDEKERCKHOVE LINOS (BE)
WEJDA MAGDALENA (BE)
GERLO SARAH (BE)
Application Number:
PCT/EP2021/056151
Publication Date:
September 16, 2021
Filing Date:
March 11, 2021
Export Citation:
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Assignee:
UNIV GENT (BE)
International Classes:
A61K35/17; A61K39/00; A61P31/18; C07K14/705; C07K14/725; C07K16/10; C07K16/28
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Claims:
Claims

1. A chimeric antigen receptor cell wherein said receptor binds to a pathogen protein expressed on a pathogen-infected cell, or, a modified T cell receptor (TCR) T cell wherein the heterodimers consisting of alpha and beta peptide chains are engineered to bind a pathogen-specific peptide expressed in the context of antigen presenting molecules on a pathogen-infected cell, characterized in that said chimeric antigen receptor cell or TCR T cell also secretes an antibody which comprises an Fc fragment and which binds said pathogen in the cell-free context.

2. A chimeric antigen receptor cell according to claim 1 wherein said pathogen protein is a HIV protein expressed on a HIV-infected cell and wherein said cell-free stage of said pathogen are free-floating HIV particles.

3. A chimeric antigen receptor cell according to claim 2 wherein said HIV protein expressed on a HIV-infected cell is an HIV envelope protein expressed on a HIV- infected CD4 positive cell.

4. A chimeric antigen receptor cell according to claims 1-3 or a modified TCR T cell according to claim 1 wherein said secreted antibody is a broadly neutralizing antibody.

5. A chimeric antigen receptor cell according to claims 1-4 wherein said cell is a CD8 positive T cell, a CD4 positive T cell or an NK cell.

6. A bicistronic vector encoding for both the chimeric antigen receptor or the modified TCR and the secreted antibody of the chimeric antigen receptor cell or of the TCR T cell as defined by claims 1-5.

7. A bicistronic vector according to claim 6 which is a lentiviral vector.

8. A bicistronic vector according to claims 6-7 comprising nucleic acid regions encoding for the following amino acid regions of the chimeric antigen receptor cell: a) an extracellular domain derived from a broadly neutralizing antibody or human CD4 receptor, a CD8 or IgG hinge region, a CD28 transmembrane domain or a 4-1BB intracellular domain or a combination of both, and a CD3zeta signaling domain, and, b) a self-cleaving peptide such as P2A orT2A, c) a human signal peptide, d) a variable heavy chain of a broadly neutralizing antibody linked to a variable light chain of a broadly neutralizing antibody, and e) a Fc region of a human IgGl, lgG2, lgG3, lgG4, lgG5, lgG6, lgG7, lgG8, lgG9, IgGlO, IgGll, lgG12, lgG13, lgG14, lgG15, lgG16, lgG17, lgG18, lgG19, or, comprising nucleic acid regions encoding for the following amino acid regions of the TCR T cell: a) an alpha chain of antigen-specific TCR, b) a self cleaving peptide such as P2A or T2A, c) a beta chain of antigen-specific TCR, d) a second self-cleaving peptide such as P2A or T2A, e) a human signal peptide, f) a variable heavy chain of a broadly neutralizing antibody linked to a variable light chain of a broadly neutralizing antibody, and g) a Fc region of a human IgGl, lgG2, lgG3, lgG4, lgG5, lgG6, lgG7, lgG8, lgG9, IgGlO, IgGll, lgG12, lgG13, lgG14, lgG15, lgG16, lgG17, lgG18, lgG19.

9. A chimeric antigen receptor cell or a modified TCR T-cell according to claims 1-5 comprising a bicistronic vector according to claims 6-7.

10. A chimeric antigen receptor cell or a modified TCR T-cell according to claims 1-5 and 9 for use as a medicament.

11. A chimeric antigen receptor cell or a modified TCR T-cell according to claims 1-5 and 9 for use to treat infections characterized by infectious agents having both a host cell associated stage and a cell-free stage inside the same host.

12. A chimeric antigen receptor for use to treat according to claim 11 wherein said infection is an infection with HIV.

13. A method to treat an infection, which is characterized by infectious agents having both a host-cell associated stage and a cell-free stage inside the same host, in a subject in need thereof comprising administering a therapeutically effective amount of a chimeric antigen receptor cell or a modified TCR T-cell according to claims 1-5 and 9.

14. A method to treat an infection according to claim 13 wherein said infection is an infection with HIV.

15. A chimeric antigen receptor cell wherein said receptor binds to an extracellular pathogen characterized in that said chimeric antigen receptor cell further secretes an antibody which binds to said extracellular pathogen. to become themselves infected with HIV virions.

17. Use of a CAR cell or a modified TCR T cell according to claim 16 which allows to use the combination of CD4+ and CD8+ CAR cells or modified TCR T cells to treat HIV.

16

Description:
Hybrid CAR and Hybrid TCR cells to treat infectious diseases

Technical field of invention

The present invention relates to the field of controlling infections where pathogens can be targeted at the host cell-associated and cell-free stages of their life cycles with edited T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR) cells which further secrete pathogen- specific antibodies having an Fc fragment. An example of such pathogen is human immunodeficiency virus (HIV). Indeed, the present invention discloses that CAR cells producing broadly neutralizing anti-HIV antibodies are capable to efficiently destroy both HIV- infected cells and non-cell associated HIV particles that become released from HIV-infected cells in the productive stage of infection. CAR-dependent killing function combined with simultaneous secretion of soluble antibody mediating neutralization of pathogen, antibody- Fc fragment mediated cellular cytotoxicity and endocytosis by antigen presenting cells following opsonization present a multiplexed approach for treatment of different infections. Background art

Chimeric Antigen Receptor or edited T Cell Receptor cells have been proposed for therapy of viral infections (1, 2). To this end, synthetic TCR or CAR encoding genes are introduced into effectorT cells in orderto reprogram their specificities towards viral antigens expressed in the context of MHC complexes or on the cell surface respectively. Such approach, however, will solely eliminate the host-cell associated pathogen, but will fail to efficiently eliminate it at the cell-free stage allowing for consecutive infection cycles. A solution to the latter problem is highly needed.

An important example of an infectious agent is Human Immunodeficiency Virus. With over 37 million people living with HIV globally, the HIV/AIDS pandemic remains one of the world's most significant public health challenges. All attempts to develop a universal curative treatment have failed, just as none of the countless tested vaccines provided protection from HIV infection. Antiretroviral therapy (ART) - the current gold standard in treatment of HIV infection fails to completely eliminate the virus from the human body. This viral persistence is mainly due to the existence of cellular reservoirs spanning CD4 positive cells of lymphoid and myeloid lineages distributed over multiple anatomical sites (3-5). These latently infected cells harbor integrated provirus that is not recognized by the host immune system and is not eliminated by ART. Therefore, therapy discontinuation results in a rapid viral rebound in all but exceptional cases. Engineered T Cell Receptor or Chimeric Antigen Receptor cells are becoming an established therapeutic approach in treatment of oncological malignancies (6). Hundreds of clinical trials worldwide are studying their potential in multiple other oncological diseases. In the HIV field, several groups have demonstrated efficient killing of infected cells by CAR-T cells (2). Unlike in cancer, however, successful treatment needs to address not only the malignant cells, but also the infectious virions they are producing. Few of the currently explored strategies address this issue. Hence most explored strategies make use of a modified TCR or CAR for direct killing effect towards the infected cells, however cell free pathogens are mostly not addressed. WO 2020/011247 to Nanjing Legend Biotech CO discloses engineered immune cells comprising a chimeric receptor targeting CCR5 or CXCR4 and a second chimeric receptor targeting CD4 wherein said immune cells are further able to secrete scFV parts of broadly neutralizing antibodies (bNAbs) in order to combat an infection with HIV. However, the latter document is silent about chimeric receptors targeting pathogen proteins instead of host cell proteins such as CCR5, CXCR4 or CD4, and -more importantly- is completely silent about which mechanism(s) might be crucial to efficiently boost host's immune responses to decrease the viral burden.

Brief description of figures

Figure 1:

Ab-secreting CAR T cells of the present invention mediate several anti-HIV functions. A. Lentiviral vectors encoding bNAb-based CAR and antibody sequences are transduced into patient-derived T cells. B. Resulting Ab-secreting CAR-T cells of the present invention: ® directly kill HIV infected cells, ® neutralize cell-free virions, mediate antibody-dependent cellular cytotoxicity (ADCC) ® and antibody-dependent cellular phagocytosis (ADCP) ® reactions. Figure 2:

Human T cells produce anti-HIV recombinant antibody. A. Histogram shows fluorescence intensity of primary human CD4+ (left panel) orCD8+ (right panel)T cells intracellularly stained with anti-lgG Fc antibody on day 3 post-transduction with control (GFP) or the antibody construct encoding (GFP-T2A-AB) lentivirus. B. Cell culture supernatant collected from the transduced cells was clarified by centrifugation and tested for the presence of antibodies with anti-human IgG ELISA.

Figure 3:

Antibodies secreted by human T cells neutralize HIV-1. Bar graph shows the level of TZM-bl cell infection as measured in a colorimetric, beta galactosidase activity assay 48 hours post infection with HIV pre-incubated with supernatants of primary T cells and normalized to no virus control. Recombinant 3BNC117 antibody was added at indicated concentration as a positive control for neutralization. Error bars indicate standard deviation between technical replicates. Figure 4:

Hybrid CAR T cells and antibodies secreted from primary T cells stop HIV replication. A) Graph indicates percentage of primary CD4+ cells infected with NL4.3 HIV virus engineered to express Heat Stable Antigen (HSA) as a marker and cocultured with autologous CD8+ T cells transduced to express constructs indicated on the figure legend. On days 3, 4 and 7 cocultured cells were stained with anti-HSA APC antibody and FACS-analyzed. B) Supernatant from day 7 cocultures performed as in A, was collected, and used for infection of indicator TZM-bl cell line. 48 hours post-infection beta-galactosidase activity marking HIV infection levels was measured. Error bars indicate standard deviation between technical replicates.

Figure 5: IgG Fc antibody domain is essential for engaging NK cells. A) Bar graph shows infectivity of HIV incubated with supernatants collected from HEK293T cells mock transfected or transfected with GFP control plasmid or anti-HIV broadly neutralizing VRC01 containing or not the IgG Fc domain. B) Bar graph shows degranulation of NK cells 7 hours post addition onto a plate coated with HIV envelope protein in presence of supernatants as described for A.

Description of invention

Most previously explored strategies -which make use of a modified TCR or CAR cells- mainly relate to direct killing effect towards the infected cells. However, the killing of cell free pathogens has hardly been addressed and -more importantly- it is not clear which immune mechanism(s) might be crucial to efficiently decrease the viral load of an infection. The present invention discloses that -besides direct neutralization of the free pathogen- antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) are crucial in order to improve the therapeutic outcome. This further means that the antibodies which are produced by the engineered cells should contain an antibody Fc fragment.

The present invention thus tackles the problem that redirected cells such as CAR cells and modified T cell receptor T cells are capable to destroy pathogen-infected cells but fail to efficiently eliminate the cell-free stage of the pathogen inside the same host.

A non-limiting list of examples of infectious agents or pathogens having both a host-cell associated stage and a cell-free stage inside the same host are for example: HIV, Herpes Simplex virus 1 and 2, Varicella Zoster virus, Human Papilloma viruses, Epstein-Barr virus, Human Cytomegalovirus, Human Herpesvirus type 8, Hepatitis B virus, Hepatitis C virus, Hepatitis Delta virus, Hepatitis E virus, Human T-cell Lymphotropic virus 1 and 2 Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium complex, Legionella pneumophila, Yersinia pestis, Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax and Plasmodium ovale.

Moreover, and in light of reports indicating direct microbicidal activity of Cytotoxic T- Lymphocytes towards extracellular pathogens (7), the modified CAR cells of the present invention, can also be used against pathogens having solely a cell-free stage. Non-limiting examples of such pathogens are: Staphylococcus aureus (including MRSA), gram negative infections like Pseudomonas Aeruginosa, Escherichia coli, Multidrug resistant Gram-negative bacteria (MDRGN), Extended Spectrum Beta-Lactamase Producing Bacteria (ESBL); fungal infections like Candida, Aspergillus and more rare variants like Mucormycosis etc. It is thus clear that the chimeric antigen receptor and the secreted Abs -which are discussed further in detail- can both bind to antigens present on cell-associated and cell-free pathogens. Indeed, the present invention discloses modified CAR cells or modified TCR T cells which are capable to secrete broadly neutralizing antibodies (bNAbs) and which neutralize free pathogens/virions. Unlike classical anti-pathogen therapy/ART focused on blocking different steps of the pathogen/viral life cycle, bNAbs can: i) bind the free pathogen/virus to inactivate or to neutralize the virus/pathogen; ii) target the virus/pathogen for phagocytosis in macrophages and dendritic cells to prime immune responses; and/or iii) attach to the surface of pathogen-infected cells/HIV-producing cells and mark them for destruction by the host's/patient's Natural Killer (NK) cells or complement system and iv) protect the modified CAR cells or modified TCR T cells from HIV infection (8) .

Hence, the present invention relates in first instance to a chimeric antigen receptor cell wherein said receptor binds to a pathogen protein expressed on a pathogen-infected cell (such as gpl20 and gp41 envelope glycoprotein in context of HIV infection or HBsAg in the context of HBV) or a modified TCR T cell wherein heterodimers consisting of alpha and beta peptide chains are engineered to bind a pathogen-specific peptide expressed in the context of antigen presenting molecules such as MHC class I or MHC class II on a pathogen-infected cell (such as HPV16 E6 protein) characterized in that said chimeric antigen receptor cell or TCR T cell also secretes an antibody which binds a pathogen in the cell-free context (for example antibody against CD4 receptor binding domain of HIV envelope glycoprotein or antibody against a major structural viral protein LI of HPV) .

More specifically, the present invention relates to a chimeric antigen receptor cell wherein said receptor binds to an HIV protein expressed on a HIV-infected cell characterized in that said chimeric antigen receptor cell also secretes an antibody which binds to free-floating HIV particles. In the infectious disease field, several groups have demonstrated efficient killing of HIV, HBV or HPV-infected cells by redirected cells such as CAR cells and modified T cell receptor T cells (1, 2, 9) so that a skilled person is taught by the art how to produce and use such cells. The terms 'a CAR cell wherein said receptor binds to an HIV protein expressed on a HIV-infected cell' thus relates to any receptor expressed by a CAR cell which binds to a protein such -as the HIV envelope protein gpl20 or gp41- present on/in the cell membrane of a HIV- infected cell resulting in the destruction or killing of said HIV-infected cell. However, the latter CAR cell receptor can also bind to free-floating HIV particles. With the term 'free-floating HIV particles' are meant 'non-cell associated HIV particles that become released from HIV-infected cells in the productive stage of infection'.

The terms 'an antibody which binds to cell-free stage of said pathogen or, more specifically to free-floating HIV particles' relates to an antibody or any functional derivative thereof or any antigen-binding fragment thereof (as long as said antibody also comprises an Fc fragment or the 'fragment crystallizable' which is the region containing only the constant regions from the heavy chain) which is not part of the above-mentioned chimeric antigen receptor and which is further characterized as being specifically directed against pathogen specific antigens such as HIV specific antigens gpl20 and/or gp41 on free-floating HIV particles but which can also bind to HIV envelope protein on the cell surface. The term "specifically directed against (or binds to) pathogens/HIV particles', when referring to said antibody, refers to a binding reaction that is determinative of the presence of a pathogen/HIV protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular pathogen/HIV protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.

According to particular embodiments, the antibodies are broadly neutralizing anti- pathogen/anti-HIV antibodies. A broadly neutralizing anti-HIV antibody means an antibody as defined above that it is capable of neutralizing multiple pathogen/HIV viral strains. Hence, the present invention relates to a chimeric antigen receptor cell or a modified TCR T cell as defined above wherein said secreted antibody is a broadly neutralizing anti-pathogen/anti-HIV antibody. In particular embodiments, combinations of multiple bNAb sequences within the CAR and/or soluble antibody domains can be used for increased potency, breadth of pathogen strain/HIV strain-coverage and to avoid escape mutations (10). The term 'Fc region of an antibody' in particular relates to the region of an antibody that mediates interaction with cell surface receptors present - among others - on natural killer cells, macrophages, dendritic cells, B lymphocytes, follicular dendritic cells, neutrophils, eosinophils, basophils, human platelets, and mast cells as well as on proteins of the complement system.

The present invention further relates to a chimeric antigen receptor cell as described above wherein said HIV protein expressed on a HIV-infected cell is an HIV envelope protein expressed on a HIV-infected cell such as a CD4 positive T cell or an astrocyte.

The present invention further relates to a chimeric antigen receptor cell as described above wherein said cell is a CD4 positive T cell, a CD8 positive T cell or a Natural Killer (NK) cell.

The present invention also relates to a bicistronic vector encoding for both the chimeric antigen receptor or modified T cell receptor T cells, and the secreted antibody of the chimeric antigen receptor cell as defined above.

More specifically, the present invention relates to a bicistronic vector as described above that can be delivered for expression in target cells by any means known in the art such as retro/lentiviral transduction (11), electroporation, microinjection (12), integration within the host genome by means of targeted gene editing with target specific endonucleases such as CRISPR/Cas9 delivery system followed by homology template-based recombination (IB) or transposon-based system such as PiggyBac or Sleeping Beauty (14).

Even more specifically, the present invention relates to a bicistronic vector as described above comprising nucleic acid regions encoding for particular amino acid regions. In the context of

CAR expressing cells the amino acid regions comprise: a) an extracellular domain derived from a broadly neutralizing anti-pathogen/anti-HIV antibody or human CD4 receptor, a CD8 or IgG hinge region, a CD28 transmembrane domain or a 4-1BB intracellular domain or a combination of both, and a CD3zeta signaling domain, and, b) a self-cleaving peptide such as P2A or T2A, c) a human signal peptide such as IL2, CD8A, CD8B, IFNG, CCL4, IL10, IL12A, IL12B, IL4, IgH, IgK or

PRSS1 (trypsin-1), d) a variable heavy chain of a broadly neutralizing antibody linked to a variable light chain of a broadly neutralizing antibody, and e) a Fc region of a human IgGl, lgG2, lgG3, lgG4, lgG5, lgG6, lgG7, lgG8, lgG9, IgGlO, IgGll, lgG12, lgG13, lgG14, lgG15, lgG16, lgG17, lgG18, lgG19 that can also be modified for improved functionality by -for example modifying the glycosylation patterns . In the context of modified TCR expressing cells the amino acid regions comprise: a) an alpha chain of antigen-specific TCR, b) a self-cleaving peptide such as P2A or T2A, c) a beta chain of antigen-specific TCR, d) a second self-cleaving peptide such as P2A or T2A, e) a human signal peptide such as IL2, CD8A, CD8B, IFNG, CCL4, IL10, IL12A, IL12B, IL4, IgH, IgK or PRSS1 (trypsin-1) f) a variable heavy chain of a broadly neutralizing antibody linked to a variable light chain of a broadly neutralizing antibody, and g) a Fc region of a human IgGl, lgG2, IgGB, lgG4, lgG5, lgG6, lgG7, lgG8, lgG9, IgGlO, IgGll, lgG12, IgGIB, lgG14, lgG15, lgG16, lgG17, lgG18, lgG19. The present invention further relates to a chimeric antigen receptor cell or modified T cell receptor T cell as described above comprising a bicistronic vector as described above.

The present invention also relates to a chimeric antigen receptor cell or modified T cell receptor T cell as described above for use as a medicament, and more particularly, for use to treat an infection such as an infection with HIV. By the term 'to treat' is meant the medical management of a patient with the intent to cure, ameliorate or stabilize progression of disease in (HIV) infected patients. It is further understood that appropriate dosage of said chimeric antigen receptor cells-, or, modified T cell receptor T cells secreting anti-pathogen/anti-HIV broadly neutralizing antibodies depends upon a number of factors within the knowledge of the skilled hematologist. The dose of these compounds will vary, for example, depending upon the identity, size, and condition of the patient being treated, however preferably 10 6 to 10 7 cells per kilogram of body weight may be administered. A pharmaceutical composition is -in general- formulated to be compatible with its intended route of administration with suitable diluents, solvents, buffers, carriers, and the like as described in detail in (15). The present invention thus relates to a method to treat an infection (such as an infection with HIV) in a subject in need thereof comprising administering a therapeutically effective amount of a chimeric antigen receptor cell or modified T cell receptor T cell as described above.

The present invention further relates to the usage of a CAR cell or a modified TCR T cell as described above to protect said cells to become themselves infected with HIV virions. Moreover, the present invention relates to the latter usage which allows to use the combination of CD4+ and CD8+ CAR cells or modified TCR T cells to treat HIV.

Indeed, the present invention relates to the usage of a chimeric antigen receptor cell or modified T cell receptor T cell as described above to prevent that the said chimeric antigen receptor cell or modified T cell receptor T cell becomes infected with HIV. More specifically, antibodies secreted by the Hybrid CAR or Hybrid TCR cells allow to provide them with HIV resistance without the need for additional modifications such as disruption of viral coreceptor expression or production of HIV entry/fusion inhibitors. T cell-secreted antibodies will serve as a "neutralizing decoy" to sequestrate HIV present in the microenvironment and preventing it from binding to the CD4 receptor physiologically expressed on the cell surface. Moreover, to ensure strong and specific binding to HIV Env, the extracellular part of the CD4 receptor can and has been used as the antigen-binding domain of CAR(2). Unfortunately, such CD4-based CARs when expressed by CD8+ T cells can render them permissive to HIV infection. The present invention allows to avoid such scenario, because the antibodies secreted in situ from Hybrid CAR construct will provide the "neutralizing decoy" effect also to CD8+ T cells that express CD4-based CAR.

Examples

1. Production of Ab-secreting T cells Primary T cells (CD3+) and their subsets (CD4+, CD8+) isolated from healthy donor's PBMCs by negative selection with antibody coated magnetic microbeads (Miltenyi Biotech) are activated and lentivi rally transduced (10) to express single chain fragment variable (scFV) sequence derived from 3BNC117 bNAb (16) fused in frame with human IgGl Fc domain derived from pFuse-hlgGl-Fc expression vector (Invivogen). The lentiviral construct additionally encodes an anti-HIV CAR or a GFP marker protein expressed from the same promoter sequence as a bicistronic mRNA with a self-cleaving peptide sequence separating the two products (Figure 1) (17). Unlike the GFP-only control, the primary CD4 and CD8 T cells transduced with the antibody-encoding construct express antibody-Fc fusion protein as verified by intracellular staining with PE-marked anti-Human IgG Fc Secondary Antibody (eBioscience), as well secrete antibody-Fc fusion protein to the cell culture medium as measured by total human IgG ELISA (ThermoFisher Scientific) and shown in Figure 2.

2. Primary T cell secreted anti-HIV antibodies neutralize HIV

Primary T cells are transduced with GFP only or GFP-T2A-3BNC117 antibody encoding constructs. Supernatants from transduced cells are collected, mixed with infectious HIV virus and used to infect indicator, TZM-bl cell line. Unlike the supernatants collected from mock- transduced or GFP-transduced controls, supernatant collected from GFP-antibody transduced cells reduces HIV infectivity as shown in Figure 3.

3. Hybrid CAR T cells eliminate HIV infected cells and T cell secreted antibodies hamper HIV replication

Lentiviral vectors encoding the Hybrid CAR construct or GFP-only and GFP-antibody controls were used to transduce primary CD8+T cells. In parallel, autologous CD4+ T cells were infected with HIV marked with murine Heat Stable Antigen. On day 3 post-infection, the CD4+ T cells were mixed with CD8+T cells transduced as described above. Three, five and seven days later, the cells in each culture condition were FACS-analyzed to measure the number HIV positive cells. Supernatant from day 7 cultures was collected and used for infection of indicator TZM- bl cells (18). Results show efficient elimination of HIV-infected cells as well as anti-viral effect of secreted antibodies (Figure 4).

4. IgG Fc containing antibodies induce degranulation of NK cells This experiment shows that only HIV-neutralizing antibodies that contain IgG Fc part can mediate additional immune functions. HEK293T cells were transfected with constructs encoding GFP control, VRC01 scFv or VRC01 scFv-lgGl Fc sequence. Supernatants from transfected cells were collected and tested for the ability to neutralize HIV and induce NK cell degranulation. As shown on Figure 5, supernatants collected from both the VRC01 scFv and VRC01 scFv-lgGl Fc transfected cells can neutralize HIV, while only the VRC01 scFv-lgGl Fc supernatant is able to induce NK cell degranulation. 5. Ab-secreting CAR T cells mediate ADCC and ADCP functions

This experiment discloses that antibody-Fc region mediated ADCC and ADCP are crucial to efficiently clear the pathogen. CD4 positive T cells isolated from healthy donors and pre infected with HIV encoding a GFP markergene are seeded in the bottom chamber of the trans- well plate and mixed with autologous NK cells or macrophages. Autologous T cells, transduced to express solely the anti-HIV CAR, solely the anti-HIV CAR-bNAb-lgG Fc and solely anti-HIV CAR-bNAb construct lacking the IgG Fc domain are added onto individual wells of a the trans well insert. The physical separation allows to decouple the CAR-mediated direct cytotoxicity from the antibody mediated functions since the secreted antibodies can migrate freely through the pores of the trans-well. Only the antibodies containing the IgG Fc domain are able to engage ADCC and ADCP functions as measured 48 hours post infection by comparing the number of HIV-GFP positive cells in the bottom chamber of the plate. Flow cytometry analyses with fluorophore-labelled anti-human CD3 and CD14 antibodies allow to quantify the number of macrophages that have engulfed the GFP+ T cells. 6. Antibodies from Hybrid CAR T cells protect them from HIV infection.

This experiment discloses that in situ secreted antibodies protect CD4-expressing Hybrid CAR T cells from HIV infection.

Primary T cells or their Hybrid CAR-expressing counterparts are inoculated with HIV encoding a GFP marker gene. Three days post-infection the cells are fixed, permeabilized and stained with fluorescently labelled RNA probes designed to bind CAR RNA transcripts. Cells are subsequently analyzed via flow cytometry and double positives (HIV eGFP and anti-RNA-fluorophore positive) are quantified in both control and Hybrid CAR T cell groups. The results demonstrate that hybrid CAR T cells are less infected with HIV virions in comparison to their primary T cell counterparts

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